BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases

Abstract

The interaction of β-lactamase inhibitory protein II (BLIP-II) with β-lactamases serves as a model system to investigate the principles underlying protein-protein interactions. Previous studies have focused on identifying the determinants of binding affinity and specificity between BLIP-II and class A β-lactamases. However, interactions between BLIP-II and other bacterial proteins have yet to be explored. Here, we provide evidence that BLIP-II binds penicillin binding protein 2a (PBP2a) from methicillin resistant Staphylococcus aureus (MRSA) with a KD in the low micromolar range. In comparison to the binding constants for the potent interaction between BLIP-II and TEM-1 β-lactamase (KD= 0.5 pM), the on-rate for BLIP-II binding PBP2a is 44,000 times slower and the off-rate is 170 times faster. Therefore, a slow association rate is a limiting factor for the potency of the interaction between BLIP-II and PBP2a. Results from alanine scanning mutagenesis of the predicted interface residues of BLIP-II indicate that charged residues on the periphery of the BLIP-II interface play a critical role for binding PBP2a in contrast to previous findings that aromatic residues at the center of the BLIP-II interface are critical for the interaction with β-lactamases. Interestingly, many of the alanine mutants at the BLIP-II interface increase kon for binding PBP2a, consistent with the association rate being a limiting factor for affinity. In summary, the results of the study reveal that BLIP-II binds PBP2a, although weakly compared to binding of β-lactamases, and provides insights into the different binding strategies used for these targets.

Exploring Additional Dimensions of Complexity in Inhibitor Design for Serine β-Lactamases: Mechanistic and Intra- and Inter-molecular Chemistry Approaches

“…Alternatively, “narrow spectrum” inhibitors should not be discounted for therapeutic purposes as they will likely cause less damage to the patients beneficial microbiome (Boucher et al, 2017 ). Another approach is to utilize naturally observed protein inhibitors of β-lactamases from Streptomyces , termed β-lactamase inhibitor proteins (BLIPs), which can be altered to modulate β-lactamase specificity (Brown et al, 2013 ; Chow et al, 2016 ; Adamski and Palzkill, 2017a , b ); peptides derived from BLIPs have been shown to have antimicrobial activity (Alaybeyoglu et al, 2015 ).…”

Deep Sequencing of a Systematic Peptide Library Reveals Conformationally‐Constrained Protein Interface Peptides that Disrupt a Protein‐Protein Interaction

“…Affinity selection : Soluble, purified TEM‐1 and KPC‐2 β‐lactamases were used for the affinity selections. The β‐lactamases were purified as previously described and used across all of the experiments [31,33,40,63] . For both TEM‐1 and KPC‐2, 100 μg was coupled to 5 mg of magnetic M‐280‐tosyl activated Dynabeads (Invitrogen, Inc.), in separate tubes.…”

“…Binding studies were performed in duplicate using BLI with purified, soluble target proteins (TEM‐1, KPC‐2, and CTX−M‐14 β‐lactamases). The β‐lactamases were purified as previously described [31,33,40,63,65] . All of the experiments were conducted utilizing an Octet RED96 instrument (ForteBio).…”

“…The β-lactamases were purified as previously described and used across all of the experiments. [31,33,40,63] For both TEM-1 and KPC-2, 100 μg was coupled to 5 mg of magnetic M-280-tosyl activated Dynabeads (Invitrogen, Inc.), in separate tubes. The manufacturer's protocol was followed to couple the target proteins to the beads in a final suspension of 500 μL volume with 10 μg/mL of β-lactamase.…”

“…The β-lactamases were purified as previously described. [31,33,40,63,65] All of the experiments were conducted utilizing an Octet RED96 instrument (ForteBio). Assays were performed at 30 °C in 96-well black plates from Greiner, with 1,000 RPM shaking conditions, in 1X Kinetics Buffer (1X PBS pH 7.4, 0.01 % BSA, 0.002 % Tween 20) from ForteBio, and with Streptavidin (SA)-coated biosensor tips from ForteBio.…”